William G. Gutheil
William G. Gutheil
Division of Pharmaceutical Sciences
UMKC School of Pharmacy
2464 Charlotte St.
Kansas City, MO 64108
Dr. Gutheil received his B.S. in Biochemistry in 1983 from Cal Poly San Louis Obispo CA, and his Ph.D. in Chemistry in 1989 from the University of Southern California. After graduation, he did Postdoctoral Research first at Harvard University in the CBBSM (Center for Biochemical & Biophysical Sciences & Medicine) under the direction of Professors Barton Holmquist and Bert Vallee, where he received training in the areas of Analytical Metallobiochemistry, Protein Biochemistry, and Molecular Biology, and was awarded an NIH Postdoctoral Fellowship. He then received additional Postdoctoral training in the Laboratory of Professor William Bachovchin at Tufts University Medical School where he received training in the areas of Bio-organic chemistry, NMR spectroscopy, and Immunochemistry. Dr. Gutheil received his first faculty appointment in 1994 in the School of Biochemistry at Meharry Medical college, a history black medical college in Nashville TN. In 2000 he was received a faculty appointment in the Division of Pharmaceutical Sciences, School of Pharmacy, at the University of Missouri in Kansas City.
Chemical Biology of Bacterial Cell Wall Biosynthesis
Our initial interest in bacterial cell wall biosynthesis was focused on the penicillin-binding proteins (PBPs). These proteins catalyze the last steps in bacterial cell wall biosynthesis, and are the targets of the Î²-lactam antibiotics. A common theme in our cell wall focused studies has been the development of improved analytical methods, and our first steps in the PBP area was to develop improved methods for the detection of D-Ala and D-Lac [1, 2], the products of PBP catalyzed reactions. These assays are very useful for in vitro studies of the low-molecular mass penicillin binding proteins, including basic enzyme characterization studies [3-5]. We have also developed peptide boronic acid inhibitors of the PBP , and in collaboration with Dr. Chris Davies of the Medical University of South Carolina we determined a crystal structure of a substrate-like peptide boronic acid complexed with E. coli PBP 5 . Subsequently, we used this structure as the basis for a large scale multivariate statistical comparison of PBP structures as a means to look for similarities and differences within the PBP active site .
The LMM PBPs are readily amenable to study since their enzyme activity is easily detectible. In contrast, the high-molecular mass PBPs, which are essential to bacterial cell viability, give generally undetectable activity. This makes their study very difficult since enzyme assays cannot be used. To address this limitation we have developed b-lactam binding assays for the HMM PBPs , including for PBP2a which is the molecular determinant of high level Î²-lactam resistance in MRSA  - a major public health threat.
During these studies, we identified D-boroAla as having good broad spectrum antibacterial activity . At this time there were no convenient analytical methods for the quantitation of early bacterial cell wall intermediates (L-Ala, D-Ala, and D-Ala-D-Ala). Using Marfey's reagent as a chiral amine derivatizing agent, and using LC-MS/MS-based separation and quantitation, we developed a very sensitive approach to quantitating the in vivo levels of these key intermediates , which we have now extended to included D-Ala-D-Lac  - the key intermediate for determining vancomycin resistance in VRE - another major public health threat.
Our current effort in this area is to develop state-of-the-art analytical approaches to all of the intermediates in the bacterial cell wall biosynthesis pathway, and to use analytical and chemical approaches to more fully understand the basis of antibiotic resistance in pathogenic bacteria, and to identify and develop novel new agents capable of countering this significant public health threat.
Numerical, Statistical, Theoretical, and Analytical Approaches to Complex Biochemical Systems
We have a longstanding interest these deeply inter-related areas. In the area of theory, we introduced the concept of hierarchical interactions in complex state systems (systems described by state parameters such as Î”Gos and extinction coefficients) [13-15]. This provides a very powerful approach for the analysis and understanding of complex systems, and we have demonstrated how this concept can be applied to the statistical analysis of complex protein-ligand binding systems such as hemoglobin oxygen binding , and to the analysis of proton equilibria in polyprotic organic and inorganic acids [17, 18].
In the area of enzyme kinetics, we introduced the first Matlab-based program for the modeling and statistical analysis of enzyme kinetics data . As we delve further into the complexities of the bacterial cell wall biosynthesis pathway, we are considering using a "Systems Biology" approach to develop a kinetic model of this pathway.
In the area of Analytical Approaches, LC-MS/MS technology has rapidly advanced to the forefront of analytical instrumentation for the Life Sciences. I set up and oversee our current School LC-MS/MS resource, currently housing two AB-Sciex QTrap instruments (2000 QTrap and 3200 QTrap). This facility is a hands on facility where students are trained in their operation, and then have complete access to the instrument to develop and refine their methods to their particular requirements. This facility is arguably the most productive core facility on the UMKC campus.
1. Gutheil, W.G., A sensitive equilibrium-based assay for D-lactate using D-lactate dehydrogenase: application to penicillin-binding protein/DD-carboxypeptidase activity assays. Analytical biochemistry, 1998. 259(1): p. 62-7.
2. Gutheil, W.G., M.E. Stefanova, and R.A. Nicholas, Fluorescent coupled enzyme assays for D-alanine: application to penicillin-binding protein and vancomycin activity assays. Analytical biochemistry, 2000. 287(2): p. 196-202.
3. Stefanova, M.E., et al., pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5. Biochim Biophys Acta, 2002. 1597(2): p. 292-300.
4. Stefanova, M.E., et al., Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities. Biochemistry, 2003. 42(49): p. 14614-25.
5. Stefanova, M.E., et al., Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4. Eur J Biochem, 2004. 271(1): p. 23-32. 6. Pechenov, A., et al., Potential transition state analogue inhibitors for the penicillin-binding proteins. Biochemistry, 2003. 42(2): p. 579-88.
7. Nicola, G., et al., Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation. Biochemistry, 2005. 44(23): p. 8207-17.
8. Stefanova, M., S. Bobba, and W.G. Gutheil, A microtiter plate-based beta-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins. Analytical biochemistry, 2010. 396(1): p. 164-6.
9. Bobba, S., et al., Microtiter plate-based assay for inhibitors of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus. Antimicrobial agents and chemotherapy, 2011. 55(6): p. 2783-7.
10. Putty, S., et al., Characterization of d-boroAla as a novel broad-spectrum antibacterial agent targeting d-Ala-d-Ala ligase. Chemical biology & drug design, 2011. 78(5): p. 757-63.
11. Jamindar, D. and W.G. Gutheil, A liquid chromatography-tandem mass spectrometry assay for Marfey's derivatives of L-Ala, D-Ala, and D-Ala-D-Ala: application to the in vivo confirmation of alanine racemase as the target of cycloserine in Escherichia coli. Analytical biochemistry, 2010. 396(1): p. 1-7.
12. Putty, S., et al., A liquid chromatography-tandem mass spectrometry assay for d-Ala-d-Lac: A key intermediate for vancomycin resistance in vancomycin-resistant enterococci. Anal Biochem, 2013. 442(2): p. 166-171.
13. Gutheil, W.G., Thermodynamic model of cooperativity in a dimeric protein: unique and independent parameters formulation. Biophys Chem, 1992. 45(2): p. 181-91.
14. Gutheil, W.G. and C.E. McKenna, Unique and independent parameters (UIP) formulation for thermodynamic models of complex protein-ligand systems. Biophys Chem, 1992. 45(2): p. 171-9.
15. Gutheil, W.G., Reformulation of thermodynamic systems with aggregation and theoretical methods for the analysis of ligand binding in proteins with monomer-multimer equilibria. Biophys Chem, 1994. 52(1): p. 83-95.
16. Gutheil, W.G., Statistical analysis of data pertaining to complex state systems by stepwise regression with reformulated parameters; application to spectroscopically monitored hemoglobin oxygen binding data. Biophys Chem, 1998. 70(3): p. 185-202.
17. Gutheil, W.G., A simple chemical example of hierarchical thermodynamic interactions: the protonation equilibria of inorganic polyprotic acids. Biophys Chem, 2000. 88(1-3): p. 35-45.
18. Gutheil, W.G., Application of hierarchical thermodynamic interactions to the protonation equilibria of organic polyprotic acids. Biophys Chem, 2000. 88(1-3): p. 119-26.
19. Gutheil, W.G., C.A. Kettner, and W.W. Bachovchin, Kinlsq: a program for fitting kinetics data with numerically integrated rate equations and its application to the analysis of slow, tight-binding inhibition data. Analytical biochemistry, 1994. 223(1): p. 13-20.